iNOS regulates activation of the NLRP3 inflammasome through the sGC/cGMP/PKG/TACE/TNF-α axis in response to cigarette smoke resulting in aortic endothelial pyroptosis and vascular dysfunction.

BACKGROUND: Cigarette smoke (CS) is associated with vascular injury and dysfunction, which may be mediated by iNOS and NLRP3. However, the exact mechanism is unknown. METHODS: iNOS-knockout and NLRP3-knockout C57BL/6 mice were exposed to air or CS. The vascular structure was examined by hematoxylin-eosin staining. The vascular tension was measured by a vascular reactivity assay. The expression of iNOS, NLRP3, caspase-1p20, IL-1ß and eNOS were measured by western blotting. Human aortic endothelial cells (HAECs) were exposed to L-NIL (iNOS inhibitor), MCC950 (NLRP3 inhibitor), ODQ (sGC inhibitor), KT5823 (PKG inhibitor) or TAPI-1 (TACE/ADAM17 inhibitor) for 1 h prior to cigarette smoke extract (CSE) treatment. The cell viability and lactate dehydrogenase activity were assessed and pyroptosis was determined by scanning electron microscopy. The mRNA expression of TNF-α, and protein expression of iNOS, active-TACE, NLRP3, caspase-1p20, IL-1ß, and eNOS were measured. RESULTS: CS resulted in shrinkage of endothelial cells, impaired aorta relaxation, reduced eNOS expression, and induced expression of iNOS, NLRP3, caspase-1p20 and IL-1ß, which could be prevented by knockdown of iNOS and NLRP3. CSE reduced cell viability, induced LDH release and pyroptosis, and promoted iNOS, NLRP3, caspase-1p20, and IL-1ß expression and reduced eNOS reduction, which could be reversed by inhibition of iNOS or NLRP3 in HAECs. Altogether, activation of the NLRP3 inflammasome by iNOS in CS-exposed HAECs may be mediated by the sGC/cGMP/PKG/TACE/TNF- α pathway. CONCLUSION: These results link iNOS to NLRP3 in CSE-stimulated HAECs through the sGC/cGMP/PKG/TACE/TNF-α pathway. The findings identify a mechanism through which iNOS and NLRP3 contribute to the pathogenesis of CS-induced pyroptosis and impaired aorta relaxation in HAECs.

Characteristic analysis of clinical coronary heart disease and coronary artery disease concerning young and middle-aged male patients.

BACKGROUND: Coronary heart disease (CHD) is a type of coronary atherosclerotic heart disease. In recent years, the incidence of CHD has been increasing annually, with an increasing number of young patients. Severe CHD may cause severe myocardial ischemia or myocardial necrosis, which in turn may cause myocardial infarction and related complications that seriously affect the life and health of the patient. AIM: To examine the coronary arteries and clinical features of young and middle-aged male patients with CHD. METHODS: From February 2019 to January 2020, 110 male CHD patients admitted to our hospital were selected as research subjects and were divided into two groups by age: middle-aged group (n = 55) and young group (n = 55). The coronary arteries and clinical features of the patients were compared. RESULTS: There were no significant differences in dyslipidemia, stroke history, high-density lipoprotein cholesterol, or triacylglycerol (P > 0.05) between the two groups. In the young group, age, diabetes, hypertension, smoking history, body mass index, family history of CHD, drinking history, fibrinogen, low-density lipoprotein cholesterol, total cholesterol, and single-vessel disease were higher than those in the middle-aged group. Correspondingly, serum uric acid, hyperuricemia, myocardial infarction, Gensini score > 50, collateral circulation, multivessel disease, double vessel disease, involvement of the right coronary artery, and involvement of the left main coronary artery were lower in the young group than in the middle-aged group. The middle-aged group mainly suffered from a high Gensini score, implicating multiple arteries, whereas the young group was mainly affected by single-vessel disease. The between-group difference was significant (P < 0.05). CONCLUSION: In CHD attacks, multiple coronary arteries are implicated in middle-aged male patients and single-vessel disease in young male patients.

Projections from Infralimbic Cortex to Paraventricular Thalamus Mediate Fear Extinction Retrieval / 神经科学通报·英文版

The paraventricular nucleus of the thalamus (PVT), which serves as a hub, receives dense projections from the medial prefrontal cortex (mPFC) and projects to the lateral division of central amygdala (CeL). The infralimbic (IL) cortex plays a crucial role in encoding and recalling fear extinction memory. Here, we found that neurons in the PVT and IL were strongly activated during fear extinction retrieval. Silencing PVT neurons inhibited extinction retrieval at recent time point (24 h after extinction), while activating them promoted extinction retrieval at remote time point (7 d after extinction), suggesting a critical role of the PVT in extinction retrieval. In the mPFC-PVT circuit, projections from IL rather than prelimbic cortex to the PVT were dominant, and disrupting the IL-PVT projection suppressed extinction retrieval. Moreover, the axons of PVT neurons preferentially projected to the CeL. Silencing the PVT-CeL circuit also suppressed extinction retrieval. Together, our findings reveal a new neural circuit for fear extinction retrieval outside the classical IL-amygdala circuit.

Identification of physicochemical properties of Scylla paramamosain allergen, arginin kinase.

BACKGROUND: Arginine kinase (AK) is expressed in a wide variety of species, including human food sources (seafood) and pests (cockroaches and moths), and has been reported as a novel allergen. However, there has been little research on the allergenicity of AK in crustaceans. In this study the physicochemical properties of AK from mud crab (Scylla paramamosain) were investigated. RESULTS: Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis, immunoblotting and inhibition enzyme-linked immunosorbent assay revealed that purified AK was unstable in thermal processing and in acid buffer. Under simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) conditions, purified AK was much more readily degraded by pepsin than by trypsin or chymotrypsin. The unpurified AK in crab myogen degraded more markedly than purified AK. In addition, in two-phase gastrointestinal digestion, AK was rapidly degraded by pepsin but resistant to trypsin and chymotrypsin digestion, while tropomyosin derived from mud crab was resistant to pepsin digestion but digested readily by trypsin or chymotrypsin. Further study of serum samples obtained from crab-allergic human patients indicated that the allergenicity of AK was markedly reduced by digestion with SGF but not SIF. CONCLUSION: AK is an important food allergen despite its unstable physicochemical properties of digestibility.

Induction of mud crab (Scylla paramamosain) tropomyosin and arginine kinase specific hypersensitivity in BALB/c mice.

BACKGROUND: Shellfish hypersensitivity is among the most common food allergies. The allergens tropomyosin (TM) and arginine kinase (AK) from mud crab (Scylla paramamosain) were purified to homogeneity. BALB/c female mice were sensitized with TM and AK by intragastric administration. Mice treated with normal saline served as the negative control (NC) group. RESULTS: Compared with NC group, mice that were treated with TM and AK developed reduced activity; meanwhile, their scratching behavior and specific-IgE level were increased. Specific-CD4 + T cells were significantly elevated in the splenocyte cultures of the mice upon TM and AK stimulation. However, compared with the positive control group (ovalbumin, OVA), there was no significant difference. The expression of IL-4 in culture cells stimulated by TM, AK, and OVA group showed significant differences from the NC group, respectively. CONCLUSION: These results indicated that a BALB/c mouse model for sensitization to TM and AK from mud crab was successfully established, and the Th2 response was observed, displaying increased immunoglobulin E levels, together with the production of interleukin 4 and allergic symptoms.

[Surgical treatment for intra-articular calcaneal fractures].

OBJECTIVE: To evaluate clinical result of surgical treatment for intra-articular calcaneal fractures using calcaneal anatomy plate. METHODS: From September 2004 to October 2009, 72 patients with intra-articular calcaneal fractures were reviewed. There were 61 males and 11 females, ranging in age from 19 to 54 years old,with an average of 39.7 years old. The course of the disease ranged from 1 to 17 days. All the patients performed X-ray and semi-coronal CT scan before and after operation. According to Sanders classification system, there were 40 cases of type III and 32 cases of type IV. All the patients were treated with lateral L-type incision and calcaneal anatomy plate. The therapeutic effects were evaluated according to the standard of calcaneal fracture of the American surgery association of foot and ankle. RESULTS: All the patients were followed up, and the duration ranged from 10 to 48 months, with a mean of 38 months. According to standard of calcaneal fracture of the American surgery association of foot and ankle, 14 patients got an excellent result, 38 good, 9 fair and 11 poor. Five patients got incision non-union. Arthritis of subtalar joint was found in 3 cases. CONCLUSION: Open reduction and internal fixation of plate is effective to get good reduction for subtalar joint, which is a good method to treat intra-articular calcaneal fracture.

Risk assessment and ecological effects of transgenic Bacillus thuringiensis crops on non-target organisms.

The application of recombinant DNA technology has resulted in many insect-resistant varieties by genetic engineering (GE). Crops expressing Cry toxins derived from Bacillus thuringiensis (Bt) have been planted worldwide, and are an effective tool for pest control. However, one ecological concern regarding the potential effects of insect-resistant GE plants on non-target organisms (NTOs) has been continually debated. In the present study, we briefly summarize the data regarding the development and commercial use of transgenic Bt varieties, elaborate on the procedure and methods for assessing the non-target effects of insect-resistant GE plants, and synthetically analyze the related research results, mostly those published between 2005 and 2010. A mass of laboratory and field studies have shown that the currently available Bt crops have no direct detrimental effects on NTOs due to their narrow spectrum of activity, and Bt crops are increasing the abundance of some beneficial insects and improving the natural control of specific pests. The use of Bt crops, such as Bt maize and Bt cotton, results in significant reductions of insecticide application and clear benefits on the environment and farmer health. Consequently, Bt crops can be a useful component of integrated pest management systems to protect the crop from targeted pests.

Purification, cloning, expression and immunological analysis of Scylla serrata arginine kinase, the crab allergen.

BACKGROUND: Although crustaceans have been reported to be one of the most common causes of IgE-mediated allergic reactions, there are no reports about the characterization and identification of arginine kinase (AK) from the mud crab (Scylla serrata) as allergen. In the present study, the purification, molecular cloning, expression and immunological analyses of the IgE allergen AK from the mud crab were investigated. RESULTS: The results showed that cloned DNA fragments of AK from the mud crab had open reading frames of 1021 bp, predicted to encode proteins with 356 amino acid residues. Sequence alignment revealed that mud crab AK shares high homology with other crustacean species. Mud crab AK gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli BL 21. 2-D electrophoresis suggested that native AK (nAK) and recombinant AK (rAK) shared the same molecular weight of 40 kDa, and the pI is 6.5 and 6.3, respectively. The nAK and rAK were further confirmed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Immunoblotting analysis and colloidal gold immunochromatographic assay (GICA) using sera from subjects with crustacean allergy confirmed that the nAK and rAK reacted positively with these sera, indicating AK is a specific allergen of mud crab. CONCLUSION: Both of purified nAK and rAK reacted positively with sera from subjects with crustacean allergy in immunoblotting and GICA analysis, indicating AK is a common allergen of mud crab. In vitro expressed AK is proposed as a source of the protein for immunological or clinical studies.

Effects of different processing methods on digestibility of Scylla paramamosain allergen (tropomyosin).

Crustacean allergy is a significant health problem around the world, and tropomyosin (TM) represents the major allergen of crustaceans. The aim of this study was to evaluate three processing methods (boiling, CUB, HPS) and identify the one method that is most effective in the degradation of TM and reduction of its IgE-binding reactivity, and make it easier to be decomposed during gastrointestinal digestion. SDS-PAGE analysis indicated that boiling had little impact on the digestive stability of TM. In contrast, combined ultrasound and boiling (CUB), and high pressure steaming (HPS) both could accelerate the digestion of TM. Similarly, western blotting and inhibition ELISA also demonstrated that the reactivity of IgG/IgE-binding of TM that was extracted from processed crab was partially decreased after treating with CUB or HPS. Among the three processing methods, HPS was the most effective method to accelerate the digestion of TM in gastrointestinal digestion, and reduce the reactivity of IgG/IgE-binding of TM. These results suggest that proper processing of crab could promote the degradation of TM in simulated gastrointestinal digestion, reduce the reactivity of IgG/IgE-binding of TM, and decrease the incidence of crab hypersensitivity in humans.

[Identification of volatiles from field cotton plant under different induction treatments].

The volatiles of field cotton plant at its squaring stage were analyzed qualitatively and quantitatively under treatments of mechanical injury (treatment A), cotton bollworm (CBW) injury (treatment B), CBW plus CBW larva injury (treatment C), and salicylic acid (SA) inducement (treatment D). The volatiles were gathered in an automatic circle system, absorbed by Tenax-TA column, and analyzed with GC-MS system. About 30 kinds of volatiles were qualitatively identified, including terpenoids, aliphatic compounds, aromatic compounds, 3-hexanone, 2-hexanone, 3-hexanol, a-pinene, beta-pinene, beta-myrcene, propenoic acid, butyl ester, acetic acid, pentyl ester, acetic acid, butyl ester, butanoic acid, 3-methyl-, ethyl ester, benzaldehyde, acetophenone and 1, 3, 6-octatriene,3 ,7-dimethyl, etc., and a total of 10 primary volatiles were detected quantitatively. The results showed that both the kinds and the contents of the volatiles were notably higher in treatments B and C than in the control. It was noteworthy that 1, 3, 6-octatriene 3, 7-dimethyl was only found in treatments B and C, i. e., it only occurred in the cotton plants injured by CBW. There were no significant differences in the kinds and contents of the volatiles between treatment A and the control. Similar to CBW injury, SA inducement also enhanced the release of cotton plant volatiles.